Assessing the genetic risk for alcohol use disorders.

The past two decades have witnessed a revolution in the field of genetics which has led to a rapid evolution in the tools and techniques available for mapping genes that contribute to genetically complex disorders such as alcohol dependence. Research in humans and in animal models of human disease has provided important new information. Among the most commonly applied approaches used in human studies are family studies, case–control studies, and genome-wide association studies. Animal models have been aimed at identifying genetic regions or individual genes involved in different aspects of alcoholism, using such approaches as quantitative trait locus analysis, genome sequencing, knockout animals, and other sophisticated molecular genetic techniques. All of these approaches have led to the identification of several genes that seem to influence the risk for alcohol dependence, which are being further analyzed. Newer studies, however, also are attempting to look at the genetic basis of alcoholism at the level of the entire genome, moving beyond the study of individual genes toward analyses of gene interactions and gene networks in the development of this devastating disease.

A ccording to the World Health Organization (http://www.who. int/substance_abuse/facts/alcohol/ en/index.html), each year alcohol causes 2.5 million (3.8 percent of total) deaths and 69.4 million (4.5 percent of total) disability-adjusted life-years (DALYs) lost to disease worldwide. Alcohol dependence (alcoholism) also is a major health problem in the United States, affecting 4 to 5 percent of the population at any given time (Grant et al. 2004); its lifetime prevalence is 12.5 percent (Hasin et al. 2007). Initially, it was unclear whether environmental factors, genetic factors, or both contributed to the risk for alcohol dependence. Early studies clearly demonstrated that genes have a role in the risk for alcohol dependence; however, it also is clear that a substantial portion of the risk for alcoholism is not genetically determined and may result from other factors, such as the environment in which a person is raised or peer influences. In addition, gene-environment interactions exist that modify alcoholism risk (for more information, see the accompanying article by Dick and Kendler,. Ever since it has become clear that genetic factors influence the risk for alcoholism, researchers have sought to identify the genes involved. However, the complex nature of alcohol dependence and related disorders has slowed progress in identifying these genes. Thus, existing data suggest that each individual genetic element has only a small influence and that it will be necessary to identify the relevant gene networks to gain a greater understanding of the contribution of genetics to alcohol abuse and dependence (for more information on genetic and molecular networks of risk, see the article by Wolen and Miles, pp. 306-317).
Historically, two major approaches have been used to determine the magnitude of the overall genetic contribution to alcohol dependence in specific populations. The first approach was to compare the similarity (i.e., concordance) for alcohol dependence among identical (i.e., monozygotic) and fraternal (i.e., dizygotic) twins-that is, these studies assessed whether if one twin had alcohol dependence the other twin did so as well. If the risk for alcoholism, at least in part, results from genetic factors, one would expect monozygotic twins, who have identical genomes, to have a higher concordance rate for alcohol dependence than dizygotic twins, who on average only share onehalf of their genomes. Studies indeed have shown higher concordance rates among monozygotic twins, confirming the presence of a genetic component in the risk for alcoholism. The second approach involved family studies to estimate the overall similarity among 1A) and analyzed DNA samples from among the nonalcoholic control subfamily members sharing differing proboth alcoholic and nonalcoholic family jects. Case-control studies often have portions of their genome (e.g., commembers at approximately 400 different been performed on small numbers of paring sons with fathers or grandfapositions within the human genome alcoholics and control subjects, limiting thers). Together, these family and twin for sequence differences. The data then their statistical power. Moreover, many studies provided convergent evidence were examined to determine whether results from these studies could not be that genetic factors account for 50 to alcohol-dependent individuals within replicated, although this inability may 60 percent of the total variance in the families shared common gene variants be caused by population differences in risk for alcohol dependence (Heath et (i.e., alleles). Finally, the investigators genetic risk. The most robust result al. 1997;McGue 1999).
reviewed the data across all families in to emerge from these studies was the On the basis of these findings, the a study to determine whether individuals demonstration that the genes involved next step was to identify specific genes with alcoholism seemed to have inher-in alcohol metabolism-that is, genes that could influence the risk for alcoited particular parts of the genome. encoding the alcohol dehydrogenase holism. Over the past three decades, Those portions of the genome that and aldehyde dehydrogenase enzymesnew developments have made it possiseem to be shared are called quantitative play important roles in the risk for ble to search for specific genes that trait loci (QTLs) and are hypothesized alcoholism (for more information, see influence the risk for alcohol depento include genes that contribute to the the article by Hurley and Edenberg, dence, both in human populations and risk for alcoholism. The QTLs can be pp. 339-344). In addition, such analyses in animal models. This article summaquite large, often covering 10 or 20 have implicated several gene pathways rizes some of these approaches used in million base pairs that may include that encode brain-signaling molecules human populations and in studies of hundreds or even thousands of genes, (i.e., neurotransmitters) and the animal models. It also describes newer of which the right one or ones (because molecules that mediate the actions approaches aimed at analyzing the more than one in the region could of opioids (i.e., opioid receptors) (for genetic basis of alcoholism at the level contribute) would need to be identi-more information, see the article by of the entire genome, thus moving fied. Although this approach was quite Borghese and Harris, pp. 345-354), beyond analyses of the roles of individchallenging, investigators were able to as well as genes in the neuroendocrine ual genes in the development of this locate several genome regions that are and neuroimmune system (see the article devastating disease. thought to include genes that contribute by Crews, pp. 355-361) and genes to the risk for alcohol dependence. regulating circadian rhythms (see the However, conclusively identifying the article by Sarkar, pp. 362-366).

Identifying genes contributing
relevant gene(s) from the many within With the rapid advances in molecular to the risk for Alcoholism each large region has proven to be genetic technology, it now is possible more difficult than anticipated.
to test the entire genome rather than The second approach, called a case-focus on individual genes suspected to Approaches in Human Populations control study, often has been used to play a role (i.e., candidate genes) or use In human studies, several strategies examine the role of a single gene in genetic variants spaced at wide intervals have been used to search for the genes complex disorders such as alcoholism. throughout the genome. Although that influence complex traits such as This strategy involves comparing two these new approaches do not test all 3 alcohol dependence, which are influ-groups of individuals: people with billion nucleotides that make up the enced by multiple genes with smaller alcohol dependence and control sub-human DNA sequence, they can test effects rather than by one or more jects who are not alcoholic, without a few thousand (or in some cases a milgenes with larger effect sizes (Edenberg regard to the participants' family histories lion or more) different positions within and Foroud 2006; also see the article (see figure 1B). In this type of study, the genome (Stranger et al. 2010). This by Agrawal and Bierut,.
investigators analyze the distribution type of study, which is called a genome-One approach, often termed linkage of sequence variants within or near a wide association study (GWASs) analysis, involves studying families gene suspected to be involved in alco-(Manolio 2010), allows a comprehensive with multiple members who have holism in the two groups, using statistical test of association across the genome, alcohol dependence. This approach methods to compare the frequencies often while comparing case and conis based on the hypothesis that genes either of different alleles or of the trol subjects. GWASs have been used might have a greater effect in these resulting genotypes between the two for many different diseases, with varyfamilies than in families with only a groups. If a certain allele contributes ing success. Several studies have now single alcoholic member. To perform to the risk for alcohol dependence, one applied this approach to begin to this type of study, researchers recruited would expect the allele and/or genotype tackle the genetics of alcohol depenhundreds of families having two or to occur more frequently among the dence (see the article by Edenberg, more alcoholic members (see figure alcohol-dependent case subjects than pp. 336-338). However, the statistical power of GWASs is a significant hurdle. Thus, very large samples are needed because most genetic variants only have small effects, and many tests need to be performed when analyzing hundreds of thousands or a million of the genetic variants known as single nucleotide polymorphisms (SNPs). In addition, the frequency of the influential alleles in a population has an impact on the sample size that is needed to detect their influence. Furthermore, it is likely that the role that a specific allele plays in the risk for alcoholism may differ among individuals, even if they all seem to have the same disorder. This can be thought of as akin to differences among patients in response to different blood pressure medications: Although the patients all have high blood pressure, the genetic makeup may determine which medication will be most effective for a given patient.

Approaches in Animals
Animal models of traits related to human alcohol use disorders can provide pertinent information about the human condition. The usefulness of this information depends on the validity of the animal model, and there is great interest in the level of consilience between the human and laboratory animal findings (for more information, see the article by Barkley-Levenson and Crabbe, pp. 325-335). As in human studies, approaches aimed at identifying QTLs have been used in animal studies of alcohol-related traits, such as alcohol consumption and sensitivity to alcohol; however, the nature of the genetic models used varies somewhat from that used in human studies. Most of the data have come from studies performed in mice, although rats have been used as well, and a recent study has compared results for rats and humans (Tabakoff et al. 2009). In one commonly used strategy, two or more strains of mice that are known to differ with regard to the alcohol-related trait under investigation are mated to each other and their offspring (called the F1 generation) then are interbred to create Each individual has two copies of this region (one inherited from their mother and one from their father). The black bar carries a version of the gene (i.e., an allele) with a variation in its sequence that increases the risk of alcoholism. Notice that in this family, all four alcoholic individuals carry one copy of the allele that increases the alcoholism risk. The individuals who are not alcohol dependent do not carry this allele. If this pattern is repeated across many families, then there is likely to be a gene that influences the risk for alcoholism in this part of the genome. B) Case-control study. The three colors represent the three possible genetic makeups (i.e., genotypes) at the marker. The cases have more individuals with the green genotype and fewer with the blue genotype, whereas the controls have more individuals with the blue genotype and fewer with the green, suggesting that the green genotype is associated with an increased risk for alcohol dependence.
a population (the F2 generation) in genes for their effects on alcohol-related gene sequences on the microarray then which the individual animals possess traits (Crabbe et al. 2006), and more can be measured, giving an indication genes from each of the originating genes have been studied since. It is of the level of gene expression for each strains in different proportions. The important to realize, however, that gene studied. A more recent modifica-F2 animals then are scored for the although a difference identified between tion of this method called RNA-seq trait studied (e.g., amount of alcohol knockout and normal mice provides (because it involves determination of ingested), and samples of their DNA evidence for a role of the gene studied, the mRNA building blocks, or RNA are analyzed (i.e., genotyped) to identhis cannot be considered definitive sequence) has allowed researchers to tify genetic differences that correspond because many interpretational issues obtain even more detailed information to differences in the alcohol trait.
are associated with this genetic tool. on gene expression (Ozsolak and Technological advances have reduced Other, more refined, gene-manipulation Milos 2011). the labor and cost associated with strategies that do not entirely eliminate Whole-genome expression profiling genotyping, making it possible to handle the activity of a gene can provide addican be used in different ways. For larger numbers of samples and to reduce tional evidence for the influence of a example, studying gene-expression genotyping intervals. Then, QTL analgiven gene. These strategies include differences between animals that are yses are performed largely as described approaches such as conditional inactisensitive or resistant to a given alcohol above for the human studies.
vation or rescue, in which gene activity effect can provide evidence for genes This approach has identified many is reduced or eliminated only under that influence sensitivity to that alcohol regions that contain QTLs; however, certain conditions that can be coneffect. Comparisons between alcoholdetermining which gene(s) in the region trolled by the researcher (e.g., Choi et exposed and non-alcohol-exposed has a significant impact has been diffial. 2002) and RNA interference, in animals provide information about cult and requires the use of additional which reduced gene expression occurs alcohol's interactions with genes and methods. One such method is finer only in a small region of the brain (e.g., gene expression. mapping using animal populations Lesscher et al. 2009;Rewal et al. 2009). Gene-expression studies also have generated by applying other breeding Animal models also can be useful in been performed with human samples, strategies (for reviews of such strategies, research directed toward understanding using postmortem brain tissue from see Milner and Buck 2010; Palmer the health consequences of alcohol alcohol-dependent individuals and and Phillips 2002). This approach has consumption, including consequences nondependent control subjects (e.g., identified several genes for which strong in different organs. Much of this research Kryger and Wilce 2010). However, evidence supports their role in alcoholis aimed at exploring how alcohol affects factors such as quality of the sample related traits (for more information, the brain and how the brain influences and incomplete history of the subject see the article by Buck et al., pp. 367different individual responses to alcohol from which the tissue was obtained 374). Currently, second-generation that may contribute to the development complicate interpretation. In animals, DNA-sequencing technologies, often of alcohol dependence. One strategy these factors can be better controlled. called next-generation sequencing, that can be used in studies of both the Finally, in addition to whole genomes are enhancing both sequencing effibrain and other tissues is called whole being examined for expression differences ciency and the detection of genetic genome expression profiling, which using RNA, DNA microarrays have been differences (Metzker 2010), and thirdexamines the activity (i.e., expression) developed for global examination of generation sequencing is emerging of thousands of genes located throughgenomic variation (Gresham et al. 2008 . Particularly in studies of the for examining the influence of single brain, however, this approach can be moving Beyond Studying one candidate genes has been the use of applied more readily to animals than gene at a Time single-gene mutant, or knockout, to humans, because a sample of the mice. For this approach, genetic engitissue to be studied is needed. This tis-Using the approaches described here, neering is used to generate a mutant sue sample is used to extract a type of researchers now have identified some gene that no longer can express the genetic material called messenger RNA genes that are thought to influence the protein it normally produces, and that (mRNA) that is generated during the risk for alcoholism in humans or to nonfunctional (knockout) gene is inserted process of gene expression. The mRNA contribute to alcohol-related traits in into the genome of test animals. Mice then is added to a microarray-that is, animal models. However, it is clear that possessing the mutated gene then are a membrane or slide on which known the genetics of alcoholism and alcoholcompared with nonmutant mice for gene sequences have been placed that related traits are complex and will differences in alcohol-related traits.
can interact with complementary RNA include not only the effect of individual Between 1996 and 2006, this approach sequences from the brain sample. The was used to study approximately 100 amount of mRNA interacting with the continued on p. 272 genetics Primer g enetics is the study of genesthe heritable information that contains the codes for proteins and other molecules which form and maintain an organism's structure and function. In most organisms, these genes are found in strands of deoxyribonucleic acid (DNA) molecules. The specific structure of the DNA (described below) ensures that the genetic information can be passed from one generation to the next, while allowing for some reorganization that results in new variations and, ultimately, evolution.
Although nearly all cells in an organism have the same set of DNA (i.e., genome), the genomes vary among organisms, ensuring that (with few exceptions) each individual is unique. The degree of this variation is a measure of how closely related two organisms are. Thus, the differences among the genomes will be smaller among members of a family than among two completely unrelated persons, and those between related species (e.g., humans and chimpanzees) will be smaller than those between more diverse species (e.g., humans and flies).
Higher organisms are made up of various tissues and organs composed of cells with a range of functions, such as nerve, blood, or muscle cells. Yet all these cells contain the same genome. To achieve the necessary variation in cell structure and function, some DNA portions are "active," or expressed, in certain cells and at particular developmental stages, leading to the production of different end products. In addition, the environment, to some extent, can influence gene expression, resulting in changes in how the organism functions in, and adapts to, its environment.

What Is DNA?
DNA is a large complex molecule constructed from building blocks called nucleotides, each of which consists of a sugar molecule (deoxyribose) attached to an organic base. There are four organic bases and, accordingly, four different nucleotides called adenosine, cytosine, guanosine, and thymidine, generally referred to by their initials A, C, G, and T. In the cell, the nucleotides are arranged as long strings, with two strings interacting at the organic bases to form a double helix. Moreover, because of the chemical structures of these bases, their interactions are highly specific, so that T nucleotides in one strand only can interact with A nucleotides on the other strand and C nucleotides only can interact with G nucleotides. As a result, the two strands are said to be complementary. This feature is the basis for the ability of the DNA to be duplicated faithfully (at least for the most part) when cells divide so that all cells in an organism carry the same DNA sequence, which also can be passed on to the next generation. However, some variations or errors (i.e., mutations) can occur during this duplication, which lead to the variations that ensure the diversity of individuals within one species and also across species.

How Is Genetic Information Converted Into Proteins?
The genetic information is encoded in the order of the nucleotides. A gene is a particular set of these nucleotides that serves as the blueprint for a specific protein. But how does the cell read this building instruction? When a protein is needed in the cell, the DNA double helix at the corresponding gene briefly splits into single strands.
This allows certain proteins that mediate specific chemical reactions (i.e., enzymes) to copy the appropriate DNA strand by bringing in new nucleotides complementary to those in the strand (see figure). This process is called transcription. However, these new nucleotides contain a different sugar (i.e., ribose) and instead of the T nucleotides use a fifth nucleotide called uracil (U). The resulting new strand, which is made up of ribose-containing A, C, G, and U nucleotides, is called a ribonucleic acid (RNA). The RNA is released from the DNA (which then "zips" back up with its complementary strand) and is processed further into a messenger RNA (mRNA) that moves as a single strand out of the nucleus into the cytoplasm. There, other enzymes can bind to the mRNA and bring protein building blocks (i.e., amino acids) together to form chains. This process is known as translation (see figure). The order in which the amino acids are assembled is determined by the sequence of nucleotides in the mRNA, with blocks of three mRNA nucleotides representing one amino acid. The sequential steps of transcription and translationfrom DNA through the intermediate mRNA to protein-are the process by which genes are expressed.

Variations Among Genes
Variations among genes, known as polymorphisms, lead to the production of different gene products (i.e., proteins) and underlie the unique characteristics of each individual. In general, any given gene is quite similar to the same gene in another organismin other words, the nucleotide sequence is conserved. For example, the genes that code for the alcoholmetabolizing enzyme alcohol dehy- drogenase are of the same size and For example, do some people pro-These probes typically are short DNA base sequence in most individuals. duce a form of alcohol dehydrogeor RNA sequences complementary to a However, small differences in the base nase that is more (or less) efficient distinctive portion of the gene of intersequence, affecting as little as one than other people? And does this est. This probe then is marked with, for nucleotide, can result in a different influence their risk of developing example, a fluorescent tag so that it can protein. In some cases, these modifialcoholism? be detected if it interacts with the correcations still allow the gene to produce To better understand how genes sponding complementary DNA a functional protein but with slight relate to diseases and other characterissequence in a sample. variations that may affect its function. tics, the National Institutes of Health This idea of using probes to identify Other changes in the nucleotide created the Human Genome Project, differences in DNA sequences can sequence, however, may result in a which set out to map the human be expanded into broad-scale studies nonfunctional or incomplete protein. genome by sequencing the entire DNA analyzing numerous gene variants The ability to conserve the sequences sequence found in human cells. across a number of organisms. These coding for important enzymes clearly (Similar mapping efforts also have been genome-wide association studies is important to cells and organisms. conducted for many other organisms.) (GWAS) allow researchers to identify One important aspect of current This project determined not only that large numbers of variants and to genetics research is the identification the DNA sequence is highly conserved relate them to different outcomesof groups within populations that among humans (about 99.5 percent is for example, those associated with carry polymorphisms in various the same in all humans) but also that different diseases, such as alcoholism. genes, resulting in gene variants numerous combinations of genetic vari-Identifying candidate genes of parcalled alleles. Identifying these ants exist that are transmitted together ticular significance, either through polymorphisms can help scientists and which are known as haplotypes.
individual probes or large-scale to better understand how the functions To identify such variations in popumethods such as GWAS, then allows of these genes and their encoded lations with large numbers of samples, more detailed study of the particular proteins differ and how they relate to researchers are using genetic probes for characteristics and expressions of certain diseases or other characteristics. specific genes with known sequences.
those genes and their role in disease.

The conversion of genetic information into protein.
continued from p. 269 It is worth noting that all these net-